Malformations involving the large arteries that exit the heart (e.g. the great vessels) are common causes of congenital cardiovascular defects (CCDs). In most circumstances, the genetic basis for these abnormalities has not been identified. During embryogenesis, the great vessels arise from six pairs of bilaterally symmetrical arteries embedded within the pharyngeal arches that undergo extensive remodeling to produce the complex pattern present at birth. Although the remodeling aspects have been extensively studied, the developmental origin of pharyngeal arch arteries (PAAs) and the genetic programs regulating their specification remain elusive. As severe great vessel defects are incompatible with life and milder deficiencies cause CCDs, our long-term goal is to elucidate the cellular source of PAA endothelium and identify genetic pathways mediating PAA establishment to potentially identify novel human disease genes. The zebrafish model organism allows for unparalleled real-time visualization and genetic dissection of PAA development. Through examination of a novel Tg(nkx2.5::ZsYellow) reporter line, we discovered ZsYellow fluorescence in PAA endothelium. This observation was surprising as nkx2.5 transcripts are not detected in this population. Based on these data, we postulate that PAA endothelium derives from an earlier nkx2.5+ cellular source in which ZsYellow fluorescence has persisted. Although completely unexplored, this hypothesis is supported by traditional nkx2.5 cre/loxP lineage tracing in mice. Thus, it is likely that nkx2.5 plays a conserved, yet heretofore unrecognized, role in great vessel establishment that warrants further investigation. Our preliminary data also demonstrate that nkx2.5 and a requisite TGF pathway component, Latent TGF Binding Protein 3 (LTBP3), are required for PAA development, but dispensable for induction of the remaining vasculature. Based on compelling preliminary data that include genetic lineage tracing, loss-of-function, and gene expression studies, I propose to test the hypothesis that LTBP3-mediated TGF signaling from the second heart field (SHF) promotes endothelial differentiation of nkx2.5-expressing PAA progenitors. Having developed new reagents for illuminating nkx2.5+ progenitors and their derivatives, our lab has the unique opportunity to directly test this hypothesis. As PAA defects cause CCDs or embryonic lethality, the proposed studies are significant for improved therapeutic approaches.